Figure 5

Proteasome inhibition using bortezomib negatively influences estrogen-induced gene regulation. (A) After pre-treatment with 50 nM bortezomib (Bort) or vehicle (ethanol, Cont) for 15 minutes, MCF7 cells were incubated with 10 nM 17β-Estradiol (E2) for 6 h before extracting total mRNA. The expression levels of estrogen target genes CXCL12, GREB1, PGR and PKIB were normalized to 28S ribosomal mRNA, graphed relative to the control sample and expressed as relative mRNA expression; mean values + SD, n = 4. (B) Chromatin conformation capture (3C) analysis of positive and negative interactions at the CXCL12 and GREB1 loci as identified by Schmidt et al.[10] and previously described[32] (upper panel). MCF7 cells, pre-treated with 50 nM bortezomib (Bort) or vehicle (ethanol, Cont) for 15 minutes, were incubated with 10 nM 17β-Estradiol (E2) for 24 h. Purified DNA samples were quantified by qPCR using a standard curve containing the respective BAC clones for CXCL12 or GREB1. The 3C template values were normalized to values from an internal control site that lies between restriction enzyme sites, graphed relative to the control sample (set to 1) and represented as normalized relative interaction; mean values + SD, n = 3.